In Maxwell""s famous Gedanken (thought) experiment, a demon operates a door between two boxes of gas at the same temperature. The demon sorts the molecules keeping the faster molecules in one box and the slower in the other, violating the basic laws of thermodynamics. This paradox has since been resolved in many different ways. Leff, H. S. and Rex, A. F. (1990), xe2x80x9cResource letter md-1: Maxwell""s demon,xe2x80x9d Am. J. Physics 58:201-209.
A similar arrangement can be used to separate particles. Consider a mixture of particles of two different sizes suspended in water in one box and pure water in the other. If the demon opens and closes the door between the boxes quickly enough so that none of the larger particles have time to diffuse through the doorway, but long enough so that some of the smaller particles have enough time to diffuse into the other box, some separation will be achieved.
Recently two experiments have been done where a spatially asymmetric potential is periodically applied in the presence of a number of Brownian particles. Faucheux, L. S. et al. (1995), xe2x80x9cOptical thermal ratchet,xe2x80x9d Physical Rev. Letters 74:1504-1507; Rousselet, J. et al. (1994), xe2x80x9cDirectional motion of Brownian particles induced by a periodic asymmetric potential,xe2x80x9d Nature 370:446-448.
This has been shown to lead to a directed motion of the particles at a rate depending on the diffusion coefficient. One experiment (Rousselet, J. et al. (1994), xe2x80x9cDirectional motion of Brownian particles induced by a periodic asymmetric potential,xe2x80x9d Nature 370:446-448) used microfabricated electrodes on a microscope slide to apply an electric field for the potential. This idea is also the subject of European Patent Publication 645169 of Mar. 29, 1995, for xe2x80x9cSeparation of particles in a fluidxe2x80x94using a saw-tooth electrode and an intermittent excitation field,xe2x80x9d Adjari, A. et al. The other experiment (Faucheux, L. S. et al. (1995), xe2x80x9cOptical thermal ratchet,xe2x80x9d Physical Rev. Letters 74:1504-1507) used a modulated optical tweezer arrangement.
Diffusion is a process which can easily be neglected at large scales, but rapidly becomes important at the microscale. The average time for a molecule to diffuse across a distance d is 2t=d2/D where D is the diffusion coefficient of the molecule. For a protein or other large molecule, diffusion is relatively slow at the macro-scale (e.g., hemoglobin with D equal to 7xc3x9710xe2x88x927 cm2/s in water at room temperature takes about 106 seconds (ten days) to diffuse across a one centimeter pipe, but about one second to diffuse across a ten micron channel).
Using tools developed by the semiconductor industry to miniaturize electronics, it is possible to fabricate intricate fluid systems with channel sizes as small as a micron. These devices can be mass-produced inexpensively and are expected to soon be in widespread use for simple analytical tests.
A process called xe2x80x9cfield-flow fractionationxe2x80x9d (FFF) has been used to separate and analyze components of a single input stream in a system not made on the microscale, but having channels small enough to produce laminar flow. Various fields, including concentration gradients, are used to produce a force perpendicular to the direction of flow to cause separation of particles in the input stream. See, e.g., Giddings, J. C., U.S. Pat. No. 3,449,938, Jun. 17, 1969, xe2x80x9cMethod for Separating and Detecting Fluid Materials;xe2x80x9d Giddings, J. C., U.S. Pat. No. 4,147,621, Apr. 3, 1979, xe2x80x9cMethod and Apparatus for Flow Field-Flow Fractionation;xe2x80x9d Giddings, J. C., U.S. Pat. No. 4,214,981, Jul. 29, 1980), xe2x80x9cSteric Field-Flow Fractionation;xe2x80x9d Giddings, J. C., et al., U.S. Pat. No. 4,250,026, Feb. 10, 1981, xe2x80x9cContinuous Steric FFF Device for The Size Separation of Particles;xe2x80x9d Giddings, J. C., et al., (1983), xe2x80x9cOutlet Stream Splitting for Sample Concentration in Field-Flow Fractionation,xe2x80x9d Separation Science and Technology 18:293-306; Giddings, J. C. (1985), xe2x80x9cOptimized Field-Flow Fractionation System Based on Dual Stream Splitters,xe2x80x9d Anal. Chem. 57:945-947; Giddings, J. C., U.S. Pat. No. 4,830,756, May 16, 1989, xe2x80x9cHigh Speed Separation of Ultra-High Molecular Weight Polymers by Hyperlayer Field-Flow Fractionation;xe2x80x9d Giddings, J. C., U.S. Pat. No. 4,141,651, Aug. 25, 1992, xe2x80x9cPinched Channel Inlet System for Reduced Relaxation Effects and Stopless Flow Injection in Field-Flow Fractionation;xe2x80x9d Giddings, J. C., U.S. Pat. No. 5,156,039 Oct. 20, 1992, xe2x80x9cProcedure for Determining the Size and Size Distribution of Particles Using Sedimentation Field-Flow Fractionation;xe2x80x9d Giddings, J. C., U.S. Pat. No. 5,193,688, Mar. 16, 1993, xe2x80x9cMethod and Apparatus for Hydrodynamic Relaxation and Sample Concentration in Field-Flow Fraction Using Permeable Wall Elements;xe2x80x9d Caldwell, K. D. et al., U.S. Pat. No. 5,240,618, Aug. 31, 1993, xe2x80x9cElectrical Field-Flow Fractionation Using Redox Couple Added to Carrier Fluid;xe2x80x9d Giddings, J. C. (1993), xe2x80x9cField-Flow Fractionation: Analysis of Macromolecular, Colloidal and Particulate Materials,xe2x80x9d Science 260:1456-1465; Wada, Y., et al., U.S. Pat. No. 5,465,849, Nov. 14, 1995, xe2x80x9cColumn and Method for Separating Particles in Accordance with their Magnetic Susceptibility.xe2x80x9d None of these references disclose the use of a separate input stream to receive particles diffused from a particle-containing input stream.
A related method for particle fractionation is the xe2x80x9cSplit Flow Thin Cellxe2x80x9d (SPLITT) process. See, e.g., Williams, P. S., et al. (1992), xe2x80x9cContinuous SPLITT Fractionation Based on a Diffusion Mechanism,xe2x80x9d Ind. Eng. Chem. Res. 31:2172-2181; and J. C. Giddings U.S. Pat. No. 5,039,426. These publications disclose devices with channels small enough to produce laminar flow, but again only provide for one inlet stream. A further U.S. patent to J. C. Giddings, U.S. Pat. No. 4,737,268, discloses a SPLITT flow cell having two inlet streams; however the second inlet stream is not an indicator stream, but rather a particle-free stream. Giddings U.S. Pat. No. 4,894,146 also discloses a SPLITT flow cell having two input streams, but no indicator stream. All these SPLITT flow methods require the presence of more than one output stream for separating various particle fractions.
None of the foregoing publications describes a channel system device capable of analyzing small particles in very small quantities of sample which may also contain larger particles, particularly larger particles capable of affecting the indicator used for the analysis. No devices or methods using indicator streams within the cell system device are described.
Microfluidic devices allow one to take advantage of diffusion as a rapid separation mechanism, which also allows for efficient and precise detection of the separated (diffused) particles. Flow behavior in microstructures differs significantly from that in the macroscopic world. Due to extremely small inertial forces in such structures, practically all flow in microstructures is laminar. This allows the movement of different layers of fluid and particles next to each other in a channel without any mixing other than diffusion. On the other hand, due to the small lateral distances in such channels, diffusion is a powerful tool to separate molecules and small particles according to their diffusion coefficients, which is generally a function of their size. A sample stream can be in laminar flow with a stream containing an indicator substance, which provides a means for detecting an analyte which has diffused from the sample stream into the indicator stream.
Weigl, B. H. and Yager, P. xe2x80x9cSilicon-Microfabricated Diffusion-Based Optical Chemical Sensor,xe2x80x9d Sensors and Actuators Bxe2x80x94xe2x80x9cEuropetrodexe2x80x9d (Conference) Apr. 2, 1996, Zurich, Switzerland; Weigl, B. H., Holl, M. A., Schutte, D., Brody, J. P., and Yager, P. xe2x80x9cDiffusion-Based Optical Chemical Detection in Silicon Flow Structures,xe2x80x9d Analytical Methods and Instrumentation, xcexcTAS 96 special edition, 1996; Weigl, B. H., van den Engh, G., Kaiser, R., Altendorf, E., and Yager, P. xe2x80x9cRapid Sequential Chemical Analysis Using Multiple Fluroescent Reporter Beads,xe2x80x9d xcexcTAS 96, Conference Proceedings, 1996; Weigl, B. H., Hixon, G. T., Kenny, M., Zebert, D., Dwinnell, S., Buj, T. and Yager, P. xe2x80x9cFluorescence Analyte Sensing in Whole Blood Based on Diffusion Separation in Silicon-Microfabricated Flow Structures, SPIE Proceedings, Feb. 9-11, 1997, J. Lakowitz (ed.), Fluorescence Sensing Technology III; and Brody, J. and Yager, P. xe2x80x9cLow Reynolds Number Micro-Fluidic Devices,xe2x80x9d Solid State Sensor and Actuator Workshop, Hilton Head, SC Jun. 2-6, 1996, all of which are incorporated by reference in their entirety, describe various devices and methods utilizing laminar flow and diffusion principles to detect the presence of and determine the concentration of various analytes in samples, e.g., whole blood.
U.S. patent application Ser. No. 08/625,808 xe2x80x9cMicrofabricated Diffusion-Based Chemical Sensor,xe2x80x9d filed Mar. 29, 1996 (now U.S. Pat. No. 5,716,852, issued on Feb. 10, 1998), U.S. patent application Ser. No. 08/829,679, xe2x80x9cMicrofabricated Diffusion-Based Chemical Sensor,xe2x80x9d filed Mar. 31, 1997 (now U.S. Pat. No. 5,972,710 issued on Oct. 26, 1999) and P.C.T. Patent Application Serial No. PCT/US 97/05245 xe2x80x9cMicrofabricated Diffusion-Based Chemical Sensor,xe2x80x9d filed Mar. 31, 1997, each of which is hereby incorporated in its entirety by reference herein, disclose a microfabricated device comprising a laminar flow channel, at least two inlets in fluid connection with the laminar flow channel for conducting into the flow channel an indicator stream and a sample stream, and an outlet. Smaller particles in the sample stream diffuse into the indicator stream, forming a detection area wherein measurements of a detectable property are made. These three applications disclose methods for determining the concentration of analytes in a sample stream by detecting the position within the laminar flow channel of analyte particles from the sample stream diffusing into the indicator stream causing a detectable change in the indicator stream. Alternatively, the position within the laminar flow channel of the region where equilibrium of diffusion of the analyte from the sample stream into the indicator stream has occurred can be used to determine analyte concentration. Additionally, information useful for determining the concentration of analyte particles in the sample stream may be obtained by providing means such as specimen channels for conducting specimen streams from the indicator stream at successive intervals along the length of the laminar flow channel. Changes in the intensity of the signals from specimen channel to specimen channel may be used to calculate the concentration of analyte particles in the original sample.
The devices of these three applications can be in fluid connection with a device such as that disclosed in U.S. patent application Ser. No. 08/534,515 xe2x80x9cSilicon Microchannel Optical Flow Cytometer,xe2x80x9d filed Sep. 27, 1995, now U.S. Pat. No. 5,726,751, issued on Mar. 10, 1998, which is incorporated in its entirety by reference herein, which allows for single-file flow of particles and which provides reflective surfaces for detection of reflected light rather than transmitted light. The devices of these three applications can alternatively be in fluid connection with a sheath flow module as disclosed in U.S. patent application Ser. No. 08/823,747 xe2x80x9cDevice and Method for 3-Dimensional Alignment of Particles in Microfabricated Flow Channels,xe2x80x9d filed Mar. 26, 1997, and now U.S. Pat. No. 6,159,739, issued on Dec. 12, 2000, which is hereby incorporated in its entirety by reference herein.
The use of quality control materials and internal standard materials is, of course, well known in the art. See L. A. Kaplan, Clinical Chemistry, 1989, 2nd ed., The C. V. Mosby Co., St. Louis, which is incorporated by reference herein to the extent that it is not inconsistent with the disclosure herein, for a discussion of previously known methods and materials for quality control, internal standards, and calibrators. The purpose of quality control of analytical testing is to make certain that each measurement performed on a sample is reliable. In general, in previously known methods of using quality control materials, as described in Kaplan""s Clinical Chemistry, p. 278, a control is measured daily (or once per shift) and can be plotted in a Levy-Jennings plot which graphs the established target average xc2x1 two standard deviations (on the y-axis) versus time, e.g., the days of the month (on the x-axis). The target value is the estimated concentration of the analyte of interest in the sample within a certain degree of accuracy, because on any given day, the measured control value will differ slightly within the specifications of the instrument, calibration, etc. The user must establish a target value for each analyte by regular laboratory procedures known in the art. However, the measured control value should be fairly constant over time. If the control values slowly drift down or up for the target value, then this indicates a trend. If the control values show a sudden jump in the values recorded from one average to another, then this indicates a shift. If a systematic bias is indicated (a change in accuracy) or a change in precision is indicated, the user must check the reagents, internal standards, and instrumentation.
As described in Kaplan""s Clinical Chemistry, the purpose of internal standards is to improve accuracy and precision as well as providing a check of quality control because for a given internal standard batch, its measured value (e.g., detectable property correlatable to concentration, as determined by any of various detecting means) should be the same over time.
In previously known methods and materials for quality control and using internal standards, quality control materials were different from those used for internal standards.
This invention relates generally to microsensors and methods for analyzing the presence and concentration of small particles in streams containing these small particles by diffusion principles. The invention is useful, for example, for analyzing blood to determine the concentration of small particles such as hydrogen, sodium or calcium ions in a stream containing cells, or for analyzing drinking water to determine its purity.
This invention provides a device for detecting the presence or determining the concentration of analyte particles in a sample stream comprising:
a) a laminar flow channel;
b) at least three inlet means in fluid connection with the laminar flow channel for respectively conducting into the laminar flow channel (1) at least one indicator stream, the indicator stream(s) preferably comprising an indicator substance, for example, a pH-sensitive dye, which indicates the presence of the analyte particles of interest by a detectable change in property when contacted with the analyte particles, (2) at least one sample stream, and (3) at least one reference stream, which may be used as a control stream, an internal standard stream, or both, or as a calibration stream;
c) wherein the laminar flow channel has a dimension (either depth and/or width) sufficiently small to allow laminar flow of the streams adjacent to each other and a length sufficient to allow analyte particles to diffuse into the indicator stream(s) to form a detection area;
d) outlet means for conducting the streams out of the laminar flow channel.
In the simplest embodiment of this invention, a single indicator stream, a single sample stream, and a single reference stream are used; however, the methods and devices of this invention may also use multiple sample and/or indicator and/or reference streams, all in laminar flow.
The methods of this invention are designed to be carried out in a device comprising microchannels of a size such that the Reynolds number for flow within the channel is below about 1. Reynolds number is the ratio of inertia to viscosity. Low Reynolds number means that inertia is essentially negligible, turbulence is essentially negligible, and the flow of the two adjacent streams is laminar, i.e., the streams do not mix except for the diffusion of particles as described above.
A method is provided herein for detecting the presence and/or determining the concentration of analyte particles in a sample stream, preferably a liquid stream, comprising:
a) conducting the sample stream into a laminar flow channel;
b) conducting an indicator stream, the indicator stream preferably comprising an indicator substance which indicates the presence of the analyte particles by a change in a detectable property when contacted with particles of the analyte, into the laminar flow channel, whereby the sample stream and the indicator stream flow in adjacent laminar flow in the channel;
c) conducting a reference stream, containing a constant concentration, including zero up to saturation, of reference particles, preferably analyte of the same type as those in the sample stream, which is a control stream, an internal standard stream, or a calibration stream, into the laminar flow channel, whereby the reference stream flows in a laminar stream adjacent to the indicator stream in said channel;
d) allowing analyte particles to diffuse into said indicator stream from said sample stream;
e) allowing reference particles to diffuse into said indicator stream from said reference stream; and
f) detecting the presence or determining the concentration of analyte and reference particles in the indicator stream.
The methods and devices of the present invention provide for running a control for each sample simultaneously with running the sample (not only once per day or once per 8-hour shift as in previously known methods of quality control). The control can also serve as an internal standard. The internal standards of this invention do not require an exogenous material, i.e., material different from the analyte of interest. Because a control of this invention can also serve as an internal standard, the methods and devices of this invention provide enhanced efficiency of fluid analysis over previously known methods and devices.